BackgroundDespite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease.Methods and ResultsActive and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of Plasmodium vivax infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe P. vivax infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease.ConclusionDifferent clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease.

BackgroundSand fly saliva has an array of pharmacological and immunomodulatory components, and immunity to saliva protects against Leishmania infection. In the present study, we have studied the immune response against Lutzomyia intermedia saliva, the main vector of Leishmania braziliensis in Brazil, and the effects of saliva pre-exposure on L. braziliensis infection employing an intradermal experimental model.Methodology/principal findingsBALB/c mice immunized with L. intermedia salivary gland sonicate (SGS) developed a saliva-specific antibody response and a cellular immune response with presence of both IFN-γ and IL-4. The inflammatory infiltrate observed in SGS-immunized mice was comprised of numerous polymorphonuclear and few mononuclear cells. Mice challenged with live L. braziliensis in the presence of saliva were not protected although lesion development was delayed. The inoculation site and draining lymph node showed continuous parasite replication and low IFN-γ to IL-4 ratio, indicating that pre-exposure to L. intermedia saliva leads to modulation of the immune response. Furthermore, in an endemic area of cutaneous leishmaniasis, patients with active lesions displayed higher levels of anti-L. intermedia saliva antibodies when compared to individuals with a positive skin test result for Leishmania.ConclusionThese results show that pre-exposure to sand fly saliva plays an important role in the outcome of cutaneous leishmaniasis, in both mice and humans. They emphasize possible hurdles in the development of vaccines based on sand fly saliva and the need to identify and select the individual salivary candidates instead of using whole salivary mixture that may favor a non-protective response.

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 10 5 parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-␥) and in the frequency of IFN-␥-secreting CD4 ؉ and CD8 ؉ T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1b, IL-23, IL-6 and TGF-b) were detected by immunohistochemistry in ML patients. IL-17 1 cells exhibited CD4 1 , CD8 1 or CD14 1 phenotypes, and numerous IL-17 1 cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.Key words: Human . Mucosal leishmaniasis . Neutrophils . Th17 IntroductionMucosal leishmaniasis (ML), a severe chronic disease caused by leishmania protozoa, remains a serious health problem in several parts of the world, including Brazil [1]. ML is at the hyperresponsive end of the spectrum of clinical diseases caused by Leishmania braziliensis [1]. Uncontrolled immune responses have been implicated in ML pathogenesis because T lymphocytes from ML patients initiate intense responses (characterised by lymphoproliferation and cytokine production) despite the low number of parasites in mucosal lesions [2][3][4]. In addition to Th1 cytokines, TGF-b and IL-6 are also produced in ML lesions, but the significance of this finding is poorly understood [5].Th17 cells participate in inflammatory responses to several human infectious agents [6,7]. IL-17, the Th17 signature cytokine, induces tissue damage mediated by neutrophil attraction and proteinase release. Neutrophil recruitment mediated by IL-17 1 cells contributes to disease progression in susceptible mouse strains infected with L. major [8]. Although the cytokine combination that leads to human Th17 differentiation and maintenance remains controversial, TGF-b and IL-6, along with IL-23 and IL-1b, have been implicated in this phenomenon [9,10].Recently in human ML, IL-17 expression has been detected determined. In this study, we expand on the observations reported by Bacellar et al. [11] by demonstrating that in addition to Th17 cells, CD8 1 and CD14 1 cells express IL-17. We also detected the presence of neutrophils expressing proteinases in tissue-damaged areas, suggesting a potential function for Th17 cells in ML lesions. Results and discussionExpression of IL-17, Th17-inducing cytokines and retinoic acid-related orphan receptor ct (RORrt) IL-17 expression was consistently higher in ML lesions (n 5 12) than in normal mucosal samples (n 5 4), as shown in Fig. 1A and B. Marked expression was detected in mononuclear cells, endothelial cells and perivascular fusiform cells. No reactivity was detected using an isotype control antibody (Fig. 1G). As for cytokines involved in IL-17 production, ML lesions presented an intense expressio...

Neutrophils are involved in the initial steps of most responses to pathogens. In the present study, we evaluated the effects of the interaction of apoptotic vs. necrotic human neutrophils on macrophage infection by Leishmania amazonensis. Phagocytosis of apoptotic, but not viable, neutrophils by Leishmania-infected macrophages led to an increase in parasite burden via a mechanism dependent on TGF-beta1 and PGE2. Conversely, infected macrophages' uptake of necrotic neutrophils induced killing of L. amazonensis. Leishmanicidal activity was dependent on TNF-alpha and neutrophilic elastase. Nitric oxide was not involved in the killing of parasites, but the interaction of necrotic neutrophils with infected macrophages resulted in high superoxide production, a process reversed by catalase, an inhibitor of reactive oxygen intermediate production. Initial events after Leishmania infection involve interactions with neutrophils; we demonstrate that phagocytosis of these cells in an apoptotic or necrotic stage can influence the outcome of infection, driving either parasite survival or destruction.

Experiments in animals verified that phlebotomine saliva enhances Leishmania infection, and vaccination with saliva prevents disease. We have shown that individuals from an endemic area of visceral leishmaniasis displayed robust antibody responses to saliva from the vector Lutzomyia longipalpis, which correlated with anti-parasite cellmediated immunity. Here, we explored human anti-saliva responses following exposure to sand flies, using an in vivo bite model in which normal volunteers were exposed four times to 30 laboratory-reared Lu. longipalpis. Following the third exposure, normal volunteers developed diverse dermatological reactions at the site of insect bite. Serum from normal volunteers displayed high levels of anti-salivary gland sonicate IgG1, IgG4 and IgE as well as several salivary gland proteins. Furthermore, following in vitro stimulation with salivary gland sonicate, there was an increased frequency of CD4 + CD25 + and CD8 + CD25 + T cells as well as IFN-c and IL-10 synthesis. Strikingly, 1 year after the first exposure, PBMC from the volunteers displayed recall IFN-c responses that correlated with a significant reduction in infection rates using a macrophage-lymphocyte autologous culture. Together, these data suggest that human immunization against sand fly saliva is feasible and recall responses are obtained even 1 year after exposure, opening perspectives for vaccination in man.

BackgroundAreas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question.Methodology and FindingsAn observational study of 636 individuals was performed in Rondônia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P<0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r = −0.6; P = 0.0003).ConclusionHBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

Background Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins.Methods and FindingsA cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11—coding for a 4.5-kDa protein—induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells.ConclusionsWe identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.